In this proposal, we describe experiments to identify and characterize how the Caenorhabditis elegans innate immune and longevity pathways contribute to protection from bacterial pathogens, particularly pathogenic E. coli (EPEC and EHECO157:H7). During the past several years, beginning with observations of Ausubel and colleagues, a variety of human pathogens, including both bacteria and fungi, have been found to kill C. elegans. This paradigm coupled with mutageneses of the bacteria and nematode has allowed dissection of both microbial virulence and host susceptibility mechanisms. Such approaches are warranted: pathogenic E. coli, for example, have extremely complex genomes comprising 1387 gains and 528 losses compared to E. coli K12 (for EHEC), and functional assays for many proposed virulence factors simply do not exist or are untenable in mammalian systems. Moreover, such approaches have provided important information about virulence in other systems. Thus many bacterial virulence factors identified in C. elegans have similar functions in mammalian or plant systems. The high degree of conservation between mammalian and C. elegans genomes raises the possibility that mechanisms of host susceptibility are likewise conserved. We have identified a means by which we can study EPEC and EHEC pathogenesis in C. elegans: under specific growth conditions, the bacteria paralyze and kill the worms via a secreted toxin. To our knowledge, ours is the only genetic system available for studying EPEC or EHEC pathogenesis. We have found that exposure to virulent or avirulent strains of EPEC or other pathogens can "condition" or "immunize" C. elegans by upregulating pathways that control innate immunity (p38 MAP kinase;PMK-1) and longevity (IGFR (DAF-2) and FOX-O(DAF-16)). Here we propose to (i) identify EPEC genes responsible for conditioning, and test their effects in mammalian pathogenesis;(ii) identify how such "warning" factors regulate genes associated with aging and innate immunity and confer protection from EPEC;and (iii) identify genes in the longevity and innate immunity pathways that confer protection against EPEC toxins. It is our expectation that these studies on EPEC pathogenesis in C. elegans will permit identification of novel bacterial virulence factors and protective mechanisms in nematode and mammalian hosts. Project Narrative: Pathogenic E. coli cause significant morbidity and mortality but understanding of the virulence mechanisms both in host and pathogen are limited. In this application, studies are proposed to explore the pathogenesis in a C. elegans model, and determine what factors in the bacteria are detected by the host and facilitate activation of protective responses.